1. Field of the Invention
This invention concerns identification, cloning and sequencing of an Arabidopsis gene encoding a transcription factor involved in response to abscisic acid (ABA) that regulates production of seed nutrient reserves and desiccation protectants. In particular, the invention concerns identification, cloning and sequencing of the Arabidopsis ABI4 locus. ABI4 is involved in regulating seed development, production of seed nutrient reserves and desiccation protectants, and also in some ABA responses of seedlings. Over expression of the ABI4 locus is involved in modification of seed quality and vegetative responses to ABA, including tolerance to stresses such as drought or salinity. The invention also concerns a method for regulation of seed viability, production of nutrient reserves and desiccation protectants in seeds, and vegetative stress tolerance.
2. Background and Related Disclosures
Seeds, fertilized ovules containing embryos which upon germination form new plants, are important as a means of carrying plant life over periods unfavorable for growth and also as a means of distributing the plant in time and in space. The yield, quality and viability of a seed lot or crop depend on its genetic background, nutrient content and ability to germinate readily and produce a normal plant even after long term storage at variable temperatures. In addition, the general health of the plants during vegetative growth will affect their ability to support the energy demands of seed set.
It would be, therefore, advantageous to have a means to regulate a seed's viability by increasing its storage-life and increasing the plant's tolerance of stresses that may be encountered during vegetative growth.
Abscisic acid (ABA) regulates many agronomically important aspects of plant development, including synthesis of seed storage proteins and lipids (Plant Sci., 61:213-217 (1988) and Plant Hormones: Physiology, Biochemistry and Molecular Biology, 2nd ed., P. J. Davies, Ed., Norwell, Mass., Kluwer Academic Publisher, 671-697 (1995)), seed desiccation tolerance and dormancy (Arabidopsis, C. Somerville and Meyerowitz, E. M., Eds., Cold Spring Harbor: Cold Spring Harbor Laboratory Press, pp. 313-334 (1994)), as well as stomatal closure (Encyclopedia of Plant Physiology, W. Haupt and M. Feinlieb, Eds., VII. Springer-Verlag, 383-441 (1979)). In addition, ABA can induce tolerance of water-, salt- and cold stress (Ann. Rev. Plant Physiology, 39: 439-473 (1988); Plant Molecular Biology, 26: 1557-1577, (1994)).
Genetic studies, especially in Arabidopsis, have identified a large number of loci involved in ABA response (Ann. Rev. Plant Physiol. Plant Mol. Biol., 49:199-222 (1998)). The ABA response mutant phenotypes include defects in storage reserve accumulation, maturation, and dormancy of seeds, and altered ABA or stress-sensitivity for control of germination inhibition, stomatal regulation, root growth, and expression of a variety of stress-induced genes. These "ABA-insensitive" (ABI) phenotypes are extensively reviewed in Arabidopsis, C. Somerville and E. Meyerowitz, Eds., (Cold Spring Harbor: Cold Spring Harbor Press, pp. 523-553 (1994)).
Most of the ABA response mutants have relatively stage-specific defects, primarily affecting either vegetative or reproductive growth. Digenic analyses indicate that these loci are likely to be acting in multiple overlapping response pathways (Plant Physiology, 94: 1172-1179, (1990), Ann. Rev. Plant Physiol. Plant Mol. Biol., 49:199-222 (1998)). The abi1 and abi2 mutants primarily affect vegetative growth, resulting in reduced tolerance of stresses such as drought. The abi3 mutants have defects in seed development, but wild-type vegetative growth. The abi3 null mutant seeds fail to become desiccation tolerant and therefore cannot endure any period of dry storage, demonstrating the extreme importance to seed viability of the ABI3-dependent signaling pathway.
To date, only 5 mutationally identified ABA response loci have been cloned. These represent only three classes of protein: two orthologous transcriptional regulators (Viviparous 1 (Vp1)) of maize as described in Plant Cell, 1: 523-532(1989) and ABA-insensitive 3 (ABI3) of Arabidopsis (Plant Cell, 4: 1251-1261 (1992)), two highly homologous members of the protein phosphatase 2C family, namely ABI1 and ABI2 of Arabidopsis (Science, 264: 1452-1455 (1994), Plant Cell, 9: 759-771 (1997)), and a farnesyl transferase encoded by Enhanced Response to ABA 1 (ERAl) of Arabidopsis (Science, 273: 1239-1241 (1996)).
The roles of ABI1, ABI2 and ABI3 relative to each other are complex. Although ABI3 is normally seed-specific and consequently is not involved in stomatal regulation, ectopic expression of ABI3 in vegetative tissue suppresses the wilty phenotype of the abi1 mutation.
Ectopic vegetative expression of ABI3 also results in ABA-inducible vegetative expression of a subset of the genes regulated by ABI3 in seeds, e.g. those encoding storage proteins (Plant Cell, 6:1567-1582 (1994)). However, there are some limitations to the efficacy of ABI3 in extending this "seed-specific" developmental program into vegetative growth: not all ABI3-regulated genes become ABA-inducible and this ABA/ABI3-dependent expression may require yet another element of the signal transduction pathway involving ABI3.
Although the genetic regulation of ABA response described previously is complex and still only poorly understood, it is clear that these ABA response loci play an important role in regulating seed quality and survival abilities.
It has now been discovered that yet another gene, the Arabidopsis ABI4 locus, is involved in important aspects of seed nutritional quality and survival properties, can regulate ABA responsiveness of vegetative tissue, and acts as an element of the signal transduction pathway of ABI3. The role of ABI4 in regulating seed quality is reflected in the fact that abi3,abi4 digenic mutants have reduced longevity where seeds remain viable for only months, rather than years, compared to their monogenic parents.
It is, therefore, a primary objective of this invention to provide and identify a new transcription factor regulating seed development, seed viability, seed quality and vegetative stress tolerance and to provide a method for modification of seed properties by manipulating seed genetic material.